Abstract/Project Summary The prevalence of endodontic pain ranges from 32-60% for irreversible pulpitis and ~60% for cases with apical periodontitis. Importantly, sex-related differences have been reported in these patients. Moreover, differences also exist in the clinical presentation with ~40% of patients with apical periodontitis experience no pain (asymptomatic patients) despite similar radiographic presentation of bone resorption as symptomatic patients. This is an important finding as the incidence of persistent pain at 1-6 years after endodontic treatment is as high as 10-12% of all patients, particularly given recent concerns about non-steroidal anti-inflammatory drug (NSAIDs) toxicity and opioid dependence and NIH calls for development of ?personalized medicine?. Therefore, mechanisms mediating inhibition of nociception in asymptomatic patients may reveal novel factors or pathways that can be activated and therefore therapeutic in symptomatic patients. We developed a clinically translational model wherein surgical biopsies collected from patients undergoing endodontic microsurgeries undergo biochemical and functional analyses. Our preliminary data demonstrates that 1) CM from symptomatic patients (with pain) evoked significant increases in [Ca2+]i in cultured TG neurons while CM from asymptomatic patients (no-pain) did not. Moreover, CM from asymptomatic patients also desensitized CAP-evoked [Ca2+]i ; and 2) significantly greater levels of ?-endorphin was detected in asymptomatic periapical lesions compared to symptomatic periapical lesions. Based on cited literature and our preliminary data, our central hypothesis is that soluble factors released from symptomatic and asymptomatic inflamed human apical tissues differentially regulate mouse TG nociceptor activities. To test this, we propose the following aims: Specific Aim #1 will determine the functional role of CM from asymptomatic patients on regulation of TG neuronal activity. We will test the hypothesis that factors released from periapical lesions from asymptomatic patients inhibit TG neuronal activity. After surgical biopsy collection, we will perform the following: 1) apply CM from asymptomatic patients to mouse TG neurons to measure in vitro calcitonin gene- related peptide (CGRP) release and [Ca2+]i under basal and stimulated conditions; 2) inject CM from asymptomatic patients in mouse vibrissal pad to evaluate in vivo spontaneous pain and mechanical allodynia; 3) determine if a non-opioid inhibitory system is also present by using naloxone; 4) if evidence for a non-opioid inhibitor/s is found, determine if it is a lipid or a peptide; and 5) conduct secondary analyses to identify potential gender-differences. Specific Aim #2 will determine the functional role of CM from symptomatic on regulation of TG neuronal activity. We will test the hypothesis that factors released from periapical lesions from symptomatic patients will activate or sensitize TG neuronal activity. After surgical biopsy collection, we will perform the following: 1) apply CM from symptomatic patients to mouse TG neurons to measure in vitro CGRP release and [Ca2+]I under basal and stimulated conditions; 2) inject CM from symptomatic patients in mouse vibrissal pad to evaluate in vivo spontaneous pain and mechanical allodynia; 3) determine if the painful soluble factor/s is a lipid or a peptide; 4) conduct secondary analyses to evaluate the effect of gender-differences. Overall, we believe this application has considerable significance: 1) There is a striking variation in pain responses in endodontic patients (Nixdorf et al. 2016; Polycarpou et al. 2005); 2) Development of a clinically translational model to understand differences in pain states and possible sex differences would provide a foundation for a future R01 on translational mechanisms of nociceptor regulation; 3) This knowledge base may extend to other non-painful inflammatory conditions (e.g., asymptomatic inflammatory prostatitis, asymptomatic inflammatory bowel disease, asymptomatic pelvic inflammatory diseases), increasing its medical significance.